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Current chimeric antigen receptor (CAR)-T manufacturing utilizes viral vectors and extensive ex vivo expansion at large central facilities leading to an exhausted CAR-T phenotype, high costs and long vein-to-vein times. While allogeneic CAR-T can reduce delays in patient treatment, they require extensive manipulation of donor cells, severe lymphodepletion and demonstrate short persistence limiting their therapeutic window. The UltraCAR-T® platform is designed to overcome these limitations by utilizing non-viral multigene delivery and a rapid, decentralized manufacturing process without ex vivo activation or expansion of T cells. Patient’s own T cells are collected and manufactured at the medical center and re-infused one day after gene transfer. Here we describe the next generation UltraCAR-T platform that addresses the inhibitory tumor microenvironment by incorporating a novel mechanism for intrinsic downregulation of one or more checkpoint inhibitor (CPI) genes. Our design achieves intrinsic CPI blockade without gene editing and is aimed at avoiding systemic toxicity and the high cost of combining CPI antibodies. Next generation UltraCAR-T cells simultaneously express CAR, membrane bound IL-15 (mbIL15), kill switch, and incorporate intrinsic CPI blockade. To illustrate the ability of this platform, we designed exemplary non-viral transposons to generate UltraCAR-T cells against multiple tumor targets incorporating intrinsic blockade of either one (PD-1) or two (PD-1 and TIGIT) CPI genes. Healthy donor T cells were transfected using the UltraPorator™ electroporation system to manufacture UltraCAR-T cells without ex vivo activation or expansion. The co-expression of CAR, mbIL15 and kill switch was confirmed by flow cytometry and western blot. Activated UltraCAR-T showed significant reduction in CPI gene expression compared to control CAR-T cells lacking the CPI blockade and did not show unintended off-target activity. Downregulation of CPI gene(s) on UltraCAR-T enhanced cytotoxicity and inflammatory cytokine production, especially at low effector to target (E:T) cell ratios, when co-cultured with PD-L1+/CD155+ tumor cells. Single-cell cytokine proteomics showed significant increase in polyfunctionality of UltraCAR-T with intrinsic downregulation of CPI gene(s). In vivo, a single infusion of receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific UltraCAR-T with intrinsic PD-1 blockade resulted in rapid expansion, an increase in preferred T cell memory (TSCM/TCM) populations, and significantly improved overall survival of ROR1+ PD-L1+ tumor bearing mice. These preclinical data highlight the improved efficacy of incorporating intrinsic CPI blockade in UltraCAR-T cells using non-viral gene delivery and an established rapid, decentralized manufacturing process. Citation Format: Tim Chan, Cheryl Bolinger, Sean Scott, Mengyan Du, Carol Poortman, Byron Koenitzer, Taranjit Athwal, Lindsey Shepard, R. Daniel Slone, Shourik Dutta, Steven Zilko, James M. Dunleavey, Giorgio Zenere, Jacques Plummer, Bernward Klocke, Christian Zinser, Shamim Ahmad, Douglas E. Brough, Rutul R. Shah, Helen Sabzevari. Incorporation of intrinsic checkpoint blockade enhances functionality of multigenic autologous UltraCAR-T® cells manufactured using non-viral gene delivery and rapid manufacturing process [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2821.

Traditional methods for chimeric antigen receptor (CAR) T manufacturing utilize viral vectors, ex vivo activation and expansion of T cells to achieve clinically relevant cell numbers, which leads to an exhausted T cell phenotype, high manufacturing costs, and treatment delays. The UltraCAR-T platform is designed to overcome these limitations using our advanced non-viral gene delivery system and a rapid, overnight manufacturing process (Blood 2019 134 (Supplement_1):2660; Blood 2020 136 (Supplement 1):17); Cancer Research 2020 80 (16Suppl):6593). UltraCAR-T cells, which express antigen specific CAR, membrane-bound IL-15 (mbIL15), and kill switch genes, are manufactured at the medical center's cGMP facility using autologous T cells and administered back to the patient only one day after gene transfer. UltraCAR-T cells are currently under clinical investigation for hematological (NCT03927261) and solid tumors (NCT03907527). Here we describe the advancement of the UltraCAR-T platform to address the inhibitory tumor microenvironment by incorporating intrinsic checkpoint blockade without the need for complex and expensive gene editing techniques. PRGN-3007, based on the next generation of the UltraCAR-T platform, is engineered to simultaneously express CAR for targeting receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is overexpressed on many hematological and solid tumors; mbIL15 for enhanced in vivo expansion and persistence; kill switch for improved safety profile; and a novel mechanism for the intrinsic blockade of PD-1 gene expression. This approach of intrinsic blockade of PD-1 expression, only on UltraCAR-T cells, is aimed to avoid systemic toxicity and high cost of checkpoint inhibitors by eliminating the need for combination treatment. PRGN-3007 is manufactured using the already established rapid and streamlined UltraCAR-T manufacturing process. PRGN-3007 was generated using multiple healthy donor T cells using multi-cistronic non-viral vector and the overnight manufacturing process. The co-expression of CAR, mbIL15 and kill switch transgenes was confirmed by flow cytometry, western blotting, and qPCR. Furthermore, PRGN-3007 showed significant reduction in PD-1 expression on UltraCAR-T cells compared to ROR1 CAR-T cells lacking PD-1 blockade (Control ROR1 CAR-T). The downregulation of PD-1 expression on PRGN-3007 resulted in enhanced ROR1-specific cytotoxicity and release of inflammatory cytokines upon co-culture with various ROR1 + PD-L1 + hematological and solid tumor cells compared to Control ROR1 CAR-T, especially at low effector to target cell ratios. Single-cell cytokine proteomics showed that the downregulation of PD-1 expression on PRGN-3007 resulted in a significantly higher number of polyfunctional CAR-T cells compared to Control ROR1 CAR-T. Expression of mbIL15 on UltraCAR-T, with or without downregulation of PD-1 expression, resulted in robust expansion in presence of ROR1 antigen, lack of autonomous expansion in absence of ROR1, and durable persistence even in absence of exogenous cytokines in vitro. Furthermore, PRGN-3007 was selectively and effectively eliminated by the kill switch activator antibody treatment. A single administration of PRGN-3007, only one day after gene transfer, effectively reduced tumor burden and significantly improved overall survival (p In summary, these preclinical data highlight the overall safety and improved efficacy of incorporating intrinsic downregulation of PD-1 expression on UltraCAR-T cells using non-viral gene delivery and the established rapid, decentralized manufacturing process. These data provide a strong rationale for the evaluation of PRGN-3007 for the treatment of ROR1 + malignancies. Figure: Overall survival in in an established model of mantle cell lymphoma in NSG mice. Tumor cells were engrafted in mice on Day 0 and treatments were administered on Day 8. Data shown is from 8 mice/group at the start of the study. * p Figure 1 Figure 1. Disclosures Chan: Precigen, Inc: Current Employment, Current equity holder in publicly-traded company. Scott: Precigen, Inc: Current Employment. Du: Precigen, Inc: Current Employment. Bolinger: Precigen, Inc: Current Employment. Poortman: Precigen, Inc: Current Employment. Shepard: Precigen, Inc: Current Employment. Koenitzer: Precigen, Inc: Current Employment. Govekung: Precigen, Inc: Current Employment. Sailor: Precigen, Inc: Current Employment. Johnson: Precigen, Inc: Current Employment. Plummer: Precigen, Inc: Current Employment. Zilko: Precigen, Inc: Current Employment. Dutta: Precigen, Inc: Current Employment. Kunchithapautham: Precigen, Inc: Current Employment. Athwal: Precigen, Inc: Current Employment. Klocke: Precigen, Inc: Current Employment. Zinser: Precigen, Inc: Current Employment. Abdeladhim: Precigen, Inc: Current Employment. Ahmad: Precigen, Inc: Current Employment; Kite, A Gilead Company: Ended employment in the past 24 months. Metenou: Precigen, Inc: Current Employment. Semnani: Precigen, Inc: Current Employment. Brough: Precigen, Inc: Current Employment, Current equity holder in publicly-traded company. Shah: Precigen: Current Employment, Current equity holder in publicly-traded company. Sabzevari: Precigen: Current Employment, Current equity holder in publicly-traded company; Kinnate BioPharma: Membership on an entity's Board of Directors or advisory committees; Compass Therapeutics: Current equity holder in publicly-traded company.

Markers in monocytes, precursors of macrophages, which are related to CAD, are largely unknown. Therefore, we aimed to identify genes in monocytes predictive of a new ischemic event in patients with CAD and/or discriminate between stable CAD and acute coronary syndrome. We included 66 patients with stable CAD, of which 24 developed a new ischemic event, and 19 patients with ACS. Circulating CD14+ monocytes were isolated with magnetic beads. RNA sequencing analysis in monocytes of patients with (n = 13) versus without (n = 11) ischemic event at follow-up and in patients with ACS (n = 12) was validated with qPCR (n = 85). MT-COI, STRN and COX10 predicted new ischemic events in CAD patients (power for separation at 1% error rate of 0.97, 0.90 and 0.77 respectively). Low MT-COI and high STRN were also related to shorter time between blood sampling and event. COX10 and ZNF484 together with MT-COI, STRN and WNK1 separated ACS completely from stable CAD patients. RNA expressions in monocytes of MT-COI, COX10, STRN, WNK1 and ZNF484 were independent of cholesterol lowering and antiplatelet treatment. They were independent of troponin T, a marker of myocardial injury. But, COX10 and ZNF484 in human plaques correlated to plaque markers of M1 macrophage polarization, reflecting vascular injury. Expression of MT-COI, COX10, STRN and WNK1, but not that of ZNF484, PBMCs paired with that in monocytes. The prospective study of relation of MT-COI, COX10, STRN, WNK1 and ZNF484 with unstable CAD is warranted. ispartof: PLOS ONE vol:14 issue:12 ispartof: location:United States status: published

In recent years, gene fusions have gained significant recognition as biomarkers. They can assist treatment decisions, are seldom found in normal tissue and are detectable through Next-generation sequencing (NGS) of the transcriptome (RNA-seq). To transform the data provided by the sequencer into robust gene fusion detection several analysis steps are needed. Usually the first step is to map the sequenced transcript fragments (RNA-seq) to a reference genome. One standard application of this approach is to estimate expression and detect variants within known genes, e.g. SNPs and indels. In case of gene fusions, however, completely novel gene structures have to be detected. Here, we describe the detection of such gene fusion events based on our comprehensive transcript annotation (ElDorado). To demonstrate the utility of our approach, we extract gene fusion candidates from eight breast cancer cell lines, which we compare to experimentally verified gene fusions. We discuss several gene fusion events, like BCAS3-BCAS4 that was only detected in the breast cancer cell line MCF7. As supporting evidence we show that gene fusions occur more frequently in copy number enriched regions (CNV analysis). In addition, we present the Transcriptome Viewer (TViewer) a tool that allows to interactively visualize gene fusions. Finally, we support detected gene fusions through literature mining based annotations and network analyses. In conclusion, we present a platform that allows detecting gene fusions and supporting them through literature knowledge as well as rich visualization capabilities. This enables scientists to better understand molecular processes, biological functions and disease associations, which will ultimately lead to better biomedical knowledge for the development of biomarkers for diagnostics and therapies.

5-methylcytosine (5mC) is converted to 5-hydroxymethylcytosine (5hmC) by the TET family of enzymes as part of a recently discovered active DNA de-methylation pathway. 5hmC plays important roles in regulation of gene expression and differentiation and has been implicated in T cell malignancies and autoimmunity. Here, we report early and widespread 5mC/5hmC remodeling during human CD4(+) T cell differentiation ex vivo at genes and cell-specific enhancers with known T cell function. We observe similar DNA de-methylation in CD4(+) memory T cells in vivo, indicating that early remodeling events persist long term in differentiated cells. Underscoring their important function, 5hmC loci were highly enriched for genetic variants associated with T cell diseases and T-cell-specific chromosomal interactions. Extensive functional validation of 22 risk variants revealed potentially pathogenic mechanisms in diabetes and multiple sclerosis. Our results support 5hmC-mediated DNA de-methylation as a key component of CD4(+) T cell biology in humans, with important implications for gene regulation and lineage commitment. Funding Agencies|Swedish Research Council; Ake Wibergs Foundation; Cancerfonden; Helmholtz Zentrum Munchen; Technische Universitat Munchen; Biotechnology and Biological Sciences Research Council (BBSRC); CEFIC; Medical Research Council (MRC)

To elucidate the role of hepatic cytochrome P450 oxidoreductase (POR) in lipid metabolism, we characterized perturbations in lipid homeostasis in a mouse model deficient in liver POR. Using an integrative approach in which transcriptomics, lipidomics, and various bioinformatic algorithms were employed, a disruption in liver lipid mobilization, oxidation, and electron transport functions were identified. Analyzing the promoters of genes in these biological processes identified common binding motifs for nuclear receptors sensitive to lipid status, while Srebp-1c binding sites were only identified in genes involved in lipid metabolism. POR-null mice had drastic increases in hepatic lipid content (diacylglycerols, triacylglycerols, phosphatidylcholine, and cholesterol esters) and a specific enrichment in n-7 and n-9 monounsaturated fatty acids (FAs). It was found that while transporters involved in peroxisomal FA oxidation were induced, mitochondrial oxidation appeared to be more tightly controlled, supporting the increase in monounsaturated FAs. Genes coding for hepatic transporters were differentially expressed, where lipid uptake was induced and efflux repressed, indicating that in the absence of hepatic POR the liver serves as a lipid reservoir. Furthermore, while significant changes in intestinal gene expression were found in POR-deficient mice, only minor changes to plasma and intestinal lipid content were observed. Thus, while liver POR plays an important role regulating gene expression and lipid metabolism locally, the hepatic deficiency of this enzyme reverberates throughout the biological system and produces a coordinated response to the low levels of circulating cholesterol and bile.

Evaluation of cancer genomes in global context is of great interest in light of changing ethnic distribution of the world population. We focused our study on men of African ancestry because of their disproportionately higher rate of prostate cancer (CaP) incidence and mortality. We present a systematic whole genome analyses, revealing alterations that differentiate African American (AA) and Caucasian American (CA) CaP genomes. We discovered a recurrent deletion on chromosome 3q13.31 centering on the LSAMP locus that was prevalent in tumors from AA men (cumulative analyses of 435 patients: whole genome sequence, 14; FISH evaluations, 101; and SNP array, 320 patients). Notably, carriers of this deletion experienced more rapid disease progression. In contrast, PTEN and ERG common driver alterations in CaP were significantly lower in AA prostate tumors compared to prostate tumors from CA. Moreover, the frequency of inter-chromosomal rearrangements was significantly higher in AA than CA tumors. These findings reveal differentially distributed somatic mutations in CaP across ancestral groups, which have implications for precision medicine strategies.

SummaryGenome-wide association studies have revealed numerous risk loci associated with diverse diseases. However, identification of disease-causing variants within association loci remains a major challenge. Divergence in gene expression due to cis-regulatory variants in noncoding regions is central to disease susceptibility. We show that integrative computational analysis of phylogenetic conservation with a complexity assessment of co-occurring transcription factor binding sites (TFBS) can identify cis-regulatory variants and elucidate their mechanistic role in disease. Analysis of established type 2 diabetes risk loci revealed a striking clustering of distinct homeobox TFBS. We identified the PRRX1 homeobox factor as a repressor of PPARG2 expression in adipose cells and demonstrate its adverse effect on lipid metabolism and systemic insulin sensitivity, dependent on the rs4684847 risk allele that triggers PRRX1 binding. Thus, cross-species conservation analysis at the level of co-occurring TFBS provides a valuable contribution to the translation of genetic association signals to disease-related molecular mechanisms.

Phototransduction machinery in vertebrate photoreceptors is contained within the membrane discs of outer segments. Daily renewal of 10% of photoreceptor outer segments requires stringent control of gene expression. Rhodopsin constitutes over 90% of the protein in rod discs, and its altered expression or transport is associated with photoreceptor dysfunction and/or death. Two cis-regulatory sequences have been identified upstream of the rhodopsin transcription start site. While the proximal promoter binds to specific transcription factors, including NRL and CRX, the rhodopsin enhancer region (RER) reportedly contributes to precise and high-level expression of rhodopsin in vivo. Here, we report the identification of RER-bound proteins by mass spectrometry. We validate the binding of NonO (p54(nrb)), a protein implicated in coupling transcription to splicing, and three NonO-interacting proteins-hnRNP M, Ywhaz and Ppp1ca. NonO and its interactors can activate rhodopsin promoter in HEK293 cells and function synergistically with NRL and CRX. DNA-binding domain of NonO is critical for rhodopsin promoter activation. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) analysis demonstrates high occupancy of NonO at rhodopsin and a subset of phototransduction genes. Furthermore, shRNA knockdown of NonO in mouse retina leads to loss of rhodopsin expression and rod cell death, which can be partially rescued by a C-terminal NonO construct. RNA-seq analysis of the NonO shRNA-treated retina revealed splicing defects and altered expression of genes, specifically those associated with phototransduction. Our studies identify an important contribution of NonO and its interacting modulator proteins in enhancing rod-specific gene expression and controlling rod homeostasis.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al., [Background]: There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance. [Results]: A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization. [Conclusions]: The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups., This work was supported by funds provided through the Gene Partnership and the Manton Center for Orphan Disease Research at Boston Children’s Hospital and the Center for Biomedical Informatics at Harvard Medical School and by generous donations in-kind of genomic sequencing services by Life Technologies (Carlsbad, CA, USA) and Complete Genomics (Mountain View, CA, USA).

INTRODUCTION: Disproportionately higher rates of prostate cancer (CaP) incidence and mortality have been reported among African American (AA) men. Although oncogenic TMPRSS2-ERG gene fusion and deletion of the PTEN tumor suppressor gene are established cancer driver gene alterations in CaP, they are known to be more prevalent among men of European ancestry. By utilizing carefully annotated specimens, this study focused on the discovery of recurrent genomic alterations in CaP of AA men in comparison to Caucasian Americans (CA). METHODS: Genomic DNA from clinically localized primary prostate tumors (Gleason 6 or 7 with primary pattern 3) and matched peripheral blood lymphocytes of seven AA and seven CA patients, were analyzed by paired-end sequencing on Illumina Genome Analyzer IIx to a depth of 30x. Following alignment to reference genome, somatic alterations on tumor DNA that include single nucleotide variants (SNVs), insertion and deletions (Indels), structural variations, copy number variations, and inter- and intra-chromosomal translocations of tumor DNA sequence were identified. To confirm prevalent genomic deletions we performed FISH analysis on a tissue microarray constructed from 42 AA and 59 CA tumor and normal samples of an independent cohort. Frequently deleted loci were further validated by analysis of TCGA CaP SNP array data from 41 AA and 279 CA prostate tumors. RESULTS: A comparative evaluation of whole genome sequences of AA and CA CaP revealed a prevalent deletion of the LSAMP locus of chromosome 3q13.31 in AA CaP. These observations were confirmed by SNP array and FISH assays in independent cohorts of specimens. AA CaP patients with LSAMP deletion showed rapid disease progression. In contrast to higher frequency of LSAMP deletion, significantly lower frequencies of PTEN and ERG alterations were noted in CaP of AA men. Furthermore, CaP genomes of AA men displayed a higher rate of inter-chromosomal rearrangements than those from CA men. CONCLUSIONS: We highlight distinct features of AA and CA CaP genomes including common CaP driver genes (TMPRSS2- ERG, PTEN) and define a novel recurrent deletion of the LSAMP locus. This study underscores the need for careful evaluations of cancer genomes in underrepresented populations in the global context with implications for precision medicine strategies. Citation Format: Albert Dobi, Gyorgy Petrovics, Hua Li, Shyh-Han Tan, Tanja Stümpel, Denise Young, Shilpa Katta, Qiyuan Li, Kai Ying, Bernward Klocke, Lakshmi Ravindranath, Indu Kohaar, Yongmei Chen, Dezső Ribli, Korbinian Grote, Hau Zou, Joseph Cheng, Clifton L. Dalgard, Shimin Zhang, István Csabai, Jacob Kagan, David Takeda, Massimo Loda, Sudhir Srivastava, Matthias Scherf, Martin Seifert, Timo Gaiser, David G. McLeod, Zoltan Szallasi, Reinhard Ebner, Thomas Werner, Isabell A. Sesterhenn, Matthew Freedman, Shiv Srivastava. LSAMP gene deletion is associated with rapid disease progression in prostate cancer of African American men. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 140.

Zn finger proteins (ZFPs) of the C2/H2 type in Xenopus laevis are encoded by a multigene family comprising several hundred members. Based upon conserved sequence features outside the Zn finger region, ZFPs can be subdivided into distinct subfamilies. Two of such subfamilies are characterized by conserved, N-terminal amino acid sequences termed the FAX and the FAR Domain. Here we present data suggesting that the zinc finger proteins of the FAR-ZFP subfamily are targets for CK II mediated phosphorylation. Expression of these proteins during oogenesis coincides with CK II activity in unfertilized eggs. Additionally, we have found that XlcOF 7.1, a member of the FAX-ZFP subfamily, is also phosphorylated by CK II. The target sites for in vitro phosphorylation are localized within the conserved N-terminal domains but not within the Zn finger regions. However, amino acid sequence comparison revealed that individual phosphoacceptor sites are not generally conserved among all members of the respective ZFP subfamilies. The relevance of a potential CK II phosphorylation for the regulation of ZFP activity in vivo is discussed.

The function of the aryl hydrocarbon receptor (AhR) in mediating the biological effect to environmental pollutants is well established. However, accumulated evidence indicates a wide range of physiological and pathological functions mediated by the AhR, suggesting the existence of endogenous AhR ligand(s). The nature of an AhR ligand remain elusive; however, it is known that the AhR is activated by several compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin or the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole. In this study, we show that physiological concentrations of tryptamine (TA) lead to induction of cytochrome P4501A1 transcription through an AhR-dependent mechanism. In addition, we show that activation of the AhR by TA requires a functional monoamino oxidase system, suggesting that TA acts as an AhR proligand possibly by converting to a high-affinity AhR ligand. Taken together, we show a possible mechanism, through which AhR signaling is activated by endogenous conversion of TA involving monoamine oxidases.

A stringent control of homeostasis is critical for functional maintenance and survival of neurons. In the mammalian retina, the basic motif leucine zipper transcription factor NRL determines rod versus cone photoreceptor cell fate and activates the expression of many rod-specific genes. Here, we report an integrated analysis of NRL-centered gene regulatory network by coupling chromatin immunoprecipitation followed by high-throughput sequencing (ChIP–Seq) data from Illumina and ABI platforms with global expression profiling and in vivo knockdown studies. We identified approximately 300 direct NRL target genes. Of these, 22 NRL targets are associated with human retinal dystrophies, whereas 95 mapped to regions of as yet uncloned retinal disease loci. In silico analysis of NRL ChIP–Seq peak sequences revealed an enrichment of distinct sets of transcription factor binding sites. Specifically, we discovered that genes involved in photoreceptor function include binding sites for both NRL and homeodomain protein CRX. Evaluation of 26 ChIP–Seq regions validated their enhancer functions in reporter assays. In vivo knockdown of 16 NRL target genes resulted in death or abnormal morphology of rod photoreceptors, suggesting their importance in maintaining retinal function. We also identified histone demethylase Kdm5b as a novel secondary node in NRL transcriptional hierarchy. Exon array analysis of flow-sorted photoreceptors in which Kdm5b was knocked down by shRNA indicated its role in regulating rod-expressed genes. Our studies identify candidate genes for retinal dystrophies, define cis-regulatory module(s) for photoreceptor-expressed genes and provide a framework for decoding transcriptional regulatory networks that dictate rod homeostasis., Author Summary The rod and cone photoreceptors in the retina are highly specialized neurons that capture photons under dim and bright light, respectively. Loss of rod photoreceptors is an early clinical manifestation in most retinal neurodegenerative diseases that eventually result in cone cell death and blindness. The transcription factor NRL is a key regulator of rod photoreceptor cell fate and gene expression. Here, we report an integrated analysis of the global transcriptional targets of NRL. We have discovered that both NRL and CRX binding sites are present in genes involved in photoreceptor function, implying their close synergistic relationship. In vivo loss-of-function analysis of 16 NRL target genes in the mouse retina resulted in death or abnormal morphology of photoreceptor cells. Furthermore, we identified histone demethylase Kdm5b as a secondary node in the NRL-centered gene regulatory network. Our studies identify NRL target genes as excellent candidates for mutation screening of patients with retinal degenerative diseases, and they provide the foundation for elucidating regulation of rod homeostasis and targets for therapeutic intervention in diseases involving photoreceptor dysfunction.

The zinc finger motif defines a large superfamily of nucleic acid binding proteins. Conserved amino acid sequence elements associated with structurally variant zinc finger clusters define subfamilies of zinc finger proteins (ZFPs). The FAR domain (Finger Associated Repeats) is a novel type of repeat element found at the amino-terminus in a subfamily of Xenopus laevis ZFPs. Northern blot analyses of three different members of the FAR subfamily (XFO 6, XFO 9-3 and XFG 68) revealed that each of these genes is transcribed during oogenesis, embryogenesis and in all investigated tissues of adult animals thereby indicating a ubiquitous distribution of transcripts. All FAR-ZFPs tested so far have specific RNA homopolymer binding activity; they associate preferentially with poly(U). The FAR repeats possess limited primary sequence homology with a sequence in the nucleolar shuttling protein NO38, within a region that contains a casein kinase II phosphorylation site.

A gene family encoding the fork head/HNF-3 domain has been identified in the South African clawed frog, Xenopus laevis. Screening of genomic DNA and gastrula stage derived cDNA libraries with a DNA fragment encoding the Drosophila fork head domain led to the isolation of a number of different clones encoding this motif. While one of the Xenopus fork head sequences, XFD-3, represents the Xenopus counterpart to rat HNF-3 beta, all other sequences encode novel types of fork head related proteins. Here we report on XFD-1, a DNA binding protein which can bind to the HNF-3 alpha target sequence. Analysis of temporal and spatial expression revealed that the gene is activated at blastula stage and that transcripts are localized in a rather thin stripe of cells invaginating the dorsal blastopore lip (organizer) during gastrulation. XFD-1 mRNA is localized within the notochord and, by the end of neurulation, is no longer detectable. In the animal cap assay the gene is activated by incubation with the vegetalizing factor (activin A) but not with bFGF.

Motivation: Promoter analysis is an essential step on the way to identify regulatory networks. A prerequisite for successful promoter analysis is the prediction of potential transcription factor binding sites (TFBS) with reasonable accuracy. The next steps in promoter analysis can be tackled only with reliable predictions, e.g. finding phylogenetically conserved patterns or identifying higher order combinations of sites in promoters of co-regulated genes. Results: We present a new version of the program MatInspector that identifies TFBS in nucleotide sequences using a large library of weight matrices. By introducing a matrix family concept, optimized thresholds, and comparative analysis, the enhanced program produces concise results avoiding redundant and false-positive matches. We describe a number of programs based on MatInspector allowing in-depth promoter analysis (DiAlignTF, FrameWorker) and targeted design of regulatory sequences (SequenceShaper). Availability: MatInspector and the other programs described here can be used online at http://www.genomatix.de/matinspector.html. Access is free after registration within certain limitations (e.g. the number of analysis per month is currently limited to 20 analyses of arbitrary sequences). Contact: cartharius@genomatix.de Supplementary information: http://www.genomatix.de/matinspector.html

Small leucine rich proteoglycans (SLRPs) constitute a family of secreted proteoglycans that are important for collagen fibrillogenesis, cellular growth, differentiation, and migration. Ten of the 13 known members of the SLRP gene family are arranged in tandem clusters on human chromosomes 1, 9, and 12. Their syntenic equivalents are on mouse chromosomes 1, 13, and 10, and rat chromosomes 13, 17, and 7. The purpose of this study was to determine whether there is evidence for control elements, which could regulate the expression of these clusters coordinately.Promoters were identified using a comparative genomics approach and Genomatix software tools. For each gene a set of human, mouse, and rat orthologous promoters was extracted from genomic sequences. Transcription factor (TF) binding site analysis combined with a literature search was performed using MatInspector and Genomatix' BiblioSphere. Inspection for the presence of interspecies conserved scaffold/matrix attachment regions (S/MARs) was performed using ElDorado annotation lists. DNAseI hypersensitivity assay, chromatin immunoprecipitation (ChIP), and transient transfection experiments were used to validate the results from bioinformatics analysis.Transcription factor binding site analysis combined with a literature search revealed co-citations between several SLRPs and TFs Runx2 and IRF1, indicating that these TFs have potential roles in transcriptional regulation of the SLRP family members. We therefore inspected all of the SLRP promoter sets for matches to IRF factors and Runx factors. Positionally conserved binding sites for the Runt domain TFs were detected in the proximal promoters of chondroadherin (CHAD) and osteomodulin (OMD) genes. Two significant models (two or more transcription factor binding sites arranged in a defined order and orientation within a defined distance range) were derived from these initial promoter sets, the HOX-Runx (homeodomain-Runt domain), and the ETS-FKHD-STAT (erythroblast transformation specific-forkhead-signal transducers and activators of transcription) models. These models were used to scan the genomic sequences of all 13 SLRP genes. The HOX-Runx model was found within the proximal promoter, exon 1, or intron 1 sequences of 11 of the 13 SLRP genes. The ETS-FKHD-STAT model was found in only 5 of these genes. Transient transfections of MG-63 cells and bovine corneal keratocytes with Runx2 isoforms confirmed the relevance of these TFs to expression of several SLRP genes. Distribution of the HOX-Runx and ETS-FKHD-STAT models within 200 kb of genomic sequence on human chromosome 9 and 500 kb sequence on chromosome 12 also were analyzed. Two regions with 3 HOX-Runx matches within a 1,000 bp window were identified on human chromosome 9; one located between OMD and osteoglycin (OGN)/mimecan genes, and the second located upstream of the putative extracellular matrix protein 2 (ECM2) promoter. The intergenic region between OMD and mimecan was shown to coincide with different patterns of DNAse I hypersensitivity sites in MG-63 and U937 cells. ChiP analysis revealed that this region binds Runx2 in U937 cells (mimecan transcript note detectable), but binds Pitx3 in MG-63 cells (expressing high level of mimecan), thereby demonstrating its functional association with mimecan expression. Upon comparing the predictions of S/MARs on the relevant chromosomal context of human chromosomes 9 and 12 and their rodent equivalents, no convincing evidence was found that the tandemly arranged genes build a chromosomal loop.Twelve of 13 known SLRP genes have at least one HOX-Runx module match in their promoter, exon 1, intron 1, or intergenic region. Although these genes are located in different clusters on different chromosomes, the common HOX-Runx module could be the basis for co-regulated expression.